The present invention relates to novel human insulin derivatives which are soluble and have a protracted profile of action, to a method of providing such derivatives, to pharmaceutical compositions containing them, and to the use of such insulin derivatives in the treatment of diabetes.
Many diabetic patients are treated with multiple daily insulin injections in a regimen comprising one or two daily injections of a protracted insulin to cover the basal requirement supplemented by bolus injections of a rapid acting insulin to cover the meal-related requirements.
Protracted insulin compositions are well known in the art. Thus, one main type of protracted insulin compositions comprises injectable aqueous suspensions of insulin crystals or amorphous insulin. In these compositions, the insulin compounds utilized typically are protamine insulin, zinc insulin or protamine zinc insulin.
Certain drawbacks are associated with the use of insulin suspensions. Thus, in order to secure an accurate dosing, the insulin particles must be suspended homogeneously by gentle shaking before a defined volume of the suspension is withdrawn from a vial or expelled from a cartridge. Also, for the storage of insulin suspensions, the temperature must be kept within more narrow limits than for insulin solutions in order to avoid lump formation or coagulation.
While it was earlier believed that protamines were non-immunogenic, it has now turned out that protamines can be immunogenic in man and that their use for medical purposes may lead to formation of antibodies (Samuel et al., Studies on the immunogenicity of protamines in humans and experimental animals by means of a micro-complement fixation test, Clin. Exp. Immunol. 33, pp. 252-260 (1978)).
Also, evidence has been found that the protamine-insulin complex is itself immunogenic (Kurtz et al., Circulating IgG antibody to protamine in patients treated with protamine-insulins. Diabetologica 25, pp. 322-324 (1983)). Therefore, with some patients the use of protracted insulin compositions containing protamines must be avoided.
Another type of protracted insulin compositions are solutions having a pH value below physiological pH from which the insulin will precipitate because of the rise in the pH value when the solution is injected. A drawback is that the solid particles of the insulin act as a local irritant causing inflammation of the tissue at the site of injection.
WO 91/12817 (Novo Nordisk A/S) discloses protracted, soluble insulin compositions comprising insulin complexes of cobalt(III). The protraction of these complexes is only intermediate and the bioavailability is reduced.
Human insulin has three primary amino groups: the N-terminal group of the A-chain and of the B-chain and the xcex5-amino group of LysB29. Several insulin derivatives which are substituted in one or more of these groups are known in the prior art. Thus, U.S. Pat. No. 3,528,960 (Eli Lilly) relates to N-carboxyaroyl insulins in which one, two or three primary ammo groups of the insulin molecule has a carboxyaroyl group. No specifically Nxcex5B29-substituted insulins are disclosed.
According to GB Patent No. 1.492.997 (Nat. Res. Dev. Corp.), it has been found that insulin with a carbamyl substitution at Nxcex5B29 has an improved profile of hypoglycaemic effect.
P laid-open patent application No. 1-254699 (Kodama Co., Ltd.) discloses insulin wherein a fatty acid is bound to the amino group of PheB1 or to the xcex5-amino group of LysB29 or to both of these. The stated purpose of the derivatisation is to obtain a pharmacologically acceptable, stable insulin preparation.
Insulins, which in the B30 position has an amino acid having at least five carbon atoms which cannot necessarily be coded for by a triplet of nucleotides, are described in JP laid-open patent application No. 57-067548 (Shionogi). The insulin analogues are claimed to be useful in the treatment of diabetes mellitus, particularly in patients who are insulin resistant due to generation of bovine or swine insulin antibodies.
U.S. Pat. No. 5,359,030 (Ekwuribe, Protein Delivery, Inc.) describes conjugation-stabilized polypeptide compositions for oral or parenteral administration comprising a polypeptide covalently coupled with a polymer including a linear polyalkylene moiety and a lipophilic moiety, said moieties being arranged so relative to each other that the polypeptide has an enhanced in vivo resistance to enzymatic degradation.
EP 511600 A2 relates i.a. to protein derivatives of the formula [protein][Z]n wherein [protein] represents a protein having n amino residues each derivable from an amino group by removal of one of its hydrogen atoms, in stead of amino groups, [Z] is a residue represented by the formula xe2x80x94COxe2x80x94Wxe2x80x94COOH wherein W is a divalent long chain hydrocarbon group which may also contain certain hetero atoms and n represents an average of the number of amide bonds between [Z] and [protein]. It is mentioned that the protein derivatives of the invention have an extremely prolonged serum half-life as compared with the proteins from which they are derived and that they exhibit no antigenicity. It is also mentioned, that insulin is one of the proteins from which derivatives according to the invention can be made, but no specific insulin derivatives are disclosed in EP 511600 nor is there any indication of a preferred [Z] or (a) preferred position(s) in which [Z] should be introduced in order to obtain useful insulin derivatives.
In the present specification, whenever the term insulin is used in a plural or a generic sense it is intended to encompass both naturally occurring insulins and insulin analogues and derivatives thereof. By xe2x80x9cinsulin derivativexe2x80x9d as used herein is meant a polypeptide having a molecular structure similar to that of human insulin including the disulphide bridges between CysA7 and CysB7 and between CysA20 and CysB19 and an internal disulphide bridge between CysA6 and CysA11, and which have insulin activity.
However, there still is a need for protracted injectable insulin compositions which are solutions and contain insulins which stay in solution after injection and possess minimal inflammatory and immunogenic properties.
One object of the present invention is to provide human insulin derivatives, with a protracted profile of action, which are soluble at physiological pH values.
Another object of the present invention is to provide a pharmaceutical composition comprising the human insulin derivatives according to the invention.
It is a further object of the invention to provide a method of making the human insulin derivatives of the invention.
Surprisingly, it has turned out that certain insulin derivatives, wherein either the amino group of the N-terminal amino acid of the B-chain has a lipophilic substituent comprising from 12 to 40 carbon atoms attached, or wherein the carboxylic acid group of the C-terminal amino acid of the B-chain has a lipophilic substituent comprising from 12 to 40 carbon atoms attached, have a protracted profile of action and are soluble at physiological pH values.
Accordingly, in its broadest aspect, the present invention relates to an insulin derivative having the following sequence: 
wherein
Xaa at position A21 is any codable amino acid except Lys, Arg and Cys;
Xaa at positions B1, B2, B3, B26, B27, B28 and B29 are, independent of each other, any codable amino acid except Cys or deleted;
Xaa at position B30 is any codable amino acid except Cys, a dipeptide comprising no Cys or Arg, a tripeptide comprising no Cys or Arg, a tetrapeptide comprising no Cys or Arg or deleted; and either the amino group of the N-terminal amino acid of the B-chain has a lipophilic group, W, attached to it which group has from 12 to 40 carbon atoms and optionally contains a group which can be negatively charged or the carboxyl group of the C-terminal amino acid of the B-chain has a lipophilic group, Z, attached to it which group has from 12 to 40 carbon atoms and optionally contains a group which can be negatively charged with the proviso that if one or more of the amino acids at position B 1, B2 and B3 is (are) deleted then the number of the N-terminal amino acid is found by counting down from CysB7 which is always assigned the number 7 and that
(a) when B1-B2-B3 is Phe-Val-Asn and A21 is Asn and B26-B27-B28-B29-B30 is Tyr-Thr-Pro-Lys-Thr (SEQ ID NO:3) or Tyr-Thr-Pro-Lys-Ala (SEQ ID NO:4), then said W or Z always contains a group which can be negatively charged; and
(b) when B29 and B30 are deleted and a group Z as defined above is present at the C-terminal amino acid of the B-chain and neither B1, B2 nor B3 is deleted then B1-B2 is different from Phe-Val or B26-B27-B28 is different from Tyr-Thr-Pro or both B1-B2 and B26-B27-B28 are different from said sequences; and
(c) when B29 and B30 are deleted and a group Z as defined above is present at the C-terminal amino acid of the B-chain and one of B1, B2 or B3 is deleted then the N-terminal amino acid of the B-chain is different from Val or the sequence B26-B27-B28 is different from Tyr-Thr-Pro or both the N-terminal amino acid of the B-chain and the sequence B26-B27-B28 are different from Val and Tyr-Thr-Pro respectively.
In a preferred embodiment, the present invention relates to an insulin derivative having the following sequence: 
wherein
Xaa at position A21 is any codable amino acid except Lys, Arg and Cys;
Xaa at positions B1, B2, B3, B26, B27, B28, B29 and B30 are, independent of each other, any codable amino acid except Cys or deleted; and either the amino group of the N-terminal amino acid of the B-chain has a lipophilic group, W, attached to it which group has from 12 to 40 carbon atoms and optionally contains a group which can be negatively charged or the carboxyl group of the C-terminal amino acid of the B-chain has a lipophilic group, Z, attached to it which group has from 12 to 40 carbon atoms and optionally contains a group which can be negatively charged with the proviso that if one or more of the amino acids at position B1, B2 and B3 is (are) deleted then the number of the N-terminal amino acid is found by counting down from CysB7 which is always assigned the number 7 and that
(a) when B1-B2-B3 is Phe-Val-Asn and A21 is Asn and B26-B27-B28-B29-B30 is Tyr-Thr-Pro-Lys-Thr or Tyr-Thr-Pro-Lys-Ala, then said W or Z always contains a group which can be negatively charged; and
(b) when B29 and B30 are deleted and a group Z as defined above is present at the C-terminal amino acid of the B-chain and neither B1, B2 nor B3 is deleted then B1-B2 is different from Phe-Val or B26-B27-B28 is different from Tyr-Thr-Pro or both B1-B2 and B26-B27-B28 are different from said sequences; and
(c) when B29 and B30 are deleted and a group Z as defined above is present at the C-terminal amino acid of the B-chain and one of B1, B2 or B3 is deleted then the N-terminal amino acid of the B-chain is different from Val or the sequence B26-B27-B28 is different from Tyr-Thr-Pro or both the N-terminal amino acid of the B-chain and the sequence B26-B27-B28 are different from Val and Tyr-Thr-Pro respectively.
When a lipophilic group, W, is attached to the xcex1-amino group of the N-terminal amino acid of the B-chain, then the bond between the xcex1-amino group and W is preferably an amide bond in which the N-terminal amino group of the B-chain constitutes the amine moiety and a group contained in W constitutes the carboxyl moiety.
When a lipophilic group, Z, is attached to the carboxyl group of the C-terminal amino acid of the B-chain, then the bond between the carboxyl group and Z is preferably an amide bond in which the C-terminal carboxyl group constitutes the carboxyl moiety and an amino group contained in Z constitutes the amine moiety.
In another preferred embodiment, the invention relates to an insulin derivative as described above wherein a lipophilic group, W, is attached to the xcex1-amino group of the N-terminal amino acid of the B-chain.
In another preferred embodiment, the invention relates to a insulin derivative as described above wherein a lipophilic group, Z, is attached to the carboxyl group of the C-terminal amino acid of the B-chain.
In another preferred embodiment, the invention relates to an insulin derivative wherein the amino acid at position A21 is selected from the group comprising Ala, Asn, Gln, Glu, Gly and Ser.
In another preferred embodiment, the invention relates to an insulin derivative wherein the amino acid at position B1 is Phe.
In another preferred embodiment, the invention relates to an insulin derivative wherein the amino acid at position B1 is deleted.
In another preferred embodiment, the invention relates to an insulin derivative wherein the amino acid at position B2 is selected from the group comprising Ala and Val.
In another preferred embodiment, the invention relates to an insulin derivative wherein the amino acid at position B3 is selected from the group comprising Asn, Gln, Glu and Thr.
In another preferred embodiment, the invention relates to an insulin derivative wherein the amino acid at position B26 is Tyr.
In another preferred embodiment, the invention relates to an insulin derivative wherein the amino acid at position B27 is Thr.
In another preferred embodiment, the invention relates to an insulin derivative wherein the amino acid at position B28 is Pro.
In another preferred embodiment, the invention relates to an insulin derivative wherein the amino acid at position B29 is Lys or Thr.
In another preferred embodiment, the invention relates to an insulin derivative wherein the amino acid at position B28 is Lys and the amino acid at position B29 is Pro.
In another preferred embodiment, the invention relates to an insulin derivative wherein the amino acid at position B30 is Thr or xcex5-acylated Lys.
In another preferred embodiment, the invention relates to an insulin derivative wherein Xaa at position 30 in SEQ ID NO:2 designates the dipeptide Thr-Lys.
In another preferred embodiment, the invention relates to an insulin derivative wherein Xaa at position 30 in SEQ ID NO:2 designates the dipeptide Gly-Lys.
In another preferred embodiment, the invention relates to an insulin derivative wherein Xaa at position 30 in SEQ ID NO:2 designates the tripeptide Glu-Ser-Lys.
In another preferred embodiment, the invention relates to an insulin derivative wherein Xaa at position 30 in SEQ ID NO:2 designates the tripeptide Thr-Gly-Lys.
In another preferred embodiment, the invention relates to an insulin derivative wherein Xaa at position 30 in SEQ ID NO:2 designates the tetrapeptide Thr-Gly-Gly-Lys.
In another preferred embodiment, the invention relates to an insulin derivative wherein Xaa at position 30 in SEQ ID NO:2 designates the tetrapeptide Thr-Glu-Gly-Lys.
In another preferred embodiment, the invention relates to an insulin derivative wherein Xaa at position 30 in SEQ ID NO:2 designates the tetrapeptide Gly-Asp-Thr-Lys.
In another preferred embodiment, the invention relates to an insulin derivative wherein the C-terminal amino acid of the B-chain is xcex5-acylated Lys and the amino acid next to the C-terminal amino acid is Gly.
In another preferred embodiment, the invention relates to an insulin derivative wherein the parent insulin is a des(B30) insulin.
In another preferred embodiment, the invention relates to an insulin derivative wherein the parent insulin is des(B30) human insulin.
In another preferred embodiment, the invention relates to an insulin derivative wherein the parent insulin is a des(B28-B30) insulin.
In another preferred embodiment, the invention relates to an insulin derivative wherein the parent insulin is a des(B27-B30) insulin.
In another preferred embodiment, the invention relates to an insulin derivative wherein the parent insulin is a des(B26-B30) insulin.
In another preferred embodiment, the invention relates to an insulin derivative wherein the amino acid at position B28 is Pro and the amino acid at position B29 is Thr.
In another preferred embodiment, the invention relates to an insulin derivative which has a group, W, as mentioned above, attached to the N-terminal xcex1-amino group of its B-chain, W being a group of the general formula CH3(CH2)nCH(COOH)NHxe2x80x94CO(CH2)2COxe2x80x94 wherein n is an integer from 9 to 15.
In another preferred embodiment, the invention relates to an insulin derivative which has a group, W, as mentioned above, attached to the N-terminal xcex1-amino group of its B-chain, W being a group of the general formula CH3(CH2)rCOxe2x80x94NHCH(COOH)(CH2)2COxe2x80x94 wherein r is an integer from 9 to 15.
In another preferred embodiment, the invention relates to an insulin derivative which has a group, W, as mentioned above, attached to the N-terminal xcex1-amino group of its B-chain, W being a group of the general formula CH3(CH2)sCOxe2x80x94NHCH((CH2)2COOH)COxe2x80x94 wherein s is an integer from 9 to 15.
In another preferred embodiment, the invention relates to an insulin derivative which has a group, Z, as mentioned above, attached to the C-terminal amino acid of its B-chain, wherein Z is a group of the general formula xe2x80x94NHCH(COOH)(CH2)4NHxe2x80x94CO(CH2)mCH3 wherein m is an integer from 8 to 18, that is, Z is a Nxcex5-acylated lysine residue.
In another preferred embodiment, the invention relates to an insulin derivative which has a group, Z, as mentioned above, attached to the C-terminal amino acid of its B-chain, wherein Z is a group of the general formula xe2x80x94NHCH(COOH)(CH2)4NHxe2x80x94COCH((CH2)2COOH)NHxe2x80x94CO(CH2)pCH3 wherein p is an integer from 10 to 16.
In another preferred embodiment, the invention relates to an insulin derivative which has a group, Z, as mentioned above, attached to the C-terminal amino acid of its B-chain, wherein Z is a group of the general formula xe2x80x94NHCH(COOH)(CH2)4NHxe2x80x94CO(CH2)2CH(COOH)NHxe2x80x94CO(CH2)qCH3 wherein q is an integer from 10 to 16.
In another preferred embodiment, the invention relates to an insulin derivative which has a group, Z, as mentioned above, which comprises a partly or completely hydrogenated cyclopentanophenanthrene skeleton.
In another preferred embodiment, the invention relates to an insulin derivative which has a group, Z, as mentioned above, which is an acylated amino acid, in particular acylated lysine.
In another preferred embodiment, the invention relates to ThrB29 human insulin with a group Z as described above attached to the C-terminal amino acid of its B-chain.
In another preferred embodiment, the invention relates to des(B28-B30) human insulin with a group Z as described above attached to the C-terminal amino acid of its B-chain.
In another preferred embodiment, the invention relates to des(B27-B30) human insulin with a group Z as described above attached to the C-terminal amino acid of its B-chain.
In another preferred embodiment, the invention relates to des(B26-B30) human insulin with a group Z as described above attached to the C-terminal amino acid of its B-chain.
In another preferred embodiment, the invention relates to the use of an insulin derivative according to the invention for the preparation of a medicament for treating diabetes.
In another preferred embodiment, the invention relates to a pharmaceutical composition for the treatment of diabetes in a patient in need of such a treatment comprising a therapeutically effective amount of an insulin derivative according to the invention together with a pharmaceutically acceptable carrier.
In another preferred embodiment, the invention relates to a pharmaceutical composition for the treatment of diabetes in a patient in need of such a treatment comprising a therapeutically effective amount of an insulin derivative according to the invention, in mixture with an insulin or an insulin analogue which has a rapid onset of action, together with a pharmaceutically acceptable carrier.
In another preferred embodiment, the invention relates to a pharmaceutical composition comprising an insulin derivative according to the invention which is soluble at physiological pH values.
In another preferred embodiment, the invention relates to a pharmaceutical composition comprising an insulin derivative according to the invention which is soluble at pH values in the interval from about 6.5 to about 8.5.
In another preferred embodiment, the invention relates to a protracted pharmaceutical composition comprising an insulin derivative according to the invention.
In another preferred embodiment, the invention relates to a pharmaceutical composition which is a solution containing from about 120 nmol/ml to about 1200 nmol/ml, preferably about 600 nmol/ml of an insulin derivative according to the invention.
In another preferred embodiment, the invention relates to a method of treating diabetes in a patient in need of such a treatment comprising administering to the patient a therapeutically effective amount of an insulin derivative according to this invention together with a pharmaceutically acceptable carrier.
In another preferred embodiment, the invention relates to a method of treating diabetes in a patient in need of such a treatment comprising administering to the patient a therapeutically effective amount of an insulin derivative according to this invention, in mixture with an insulin or an insulin analogue which has a rapid onset of action, together with a pharmaceutically acceptable carrier.
Examples of preferred insulin derivatives according to the present invention are the following:
(Nexcex5B30-tetradecanoyl) ThrB29, LysB30 human insulin,
(Nxcex5B28-tetradecanoyl) LysB28 des(B29,B30) human insulin,
(Nxcex5B27-tetradecanoyl) LysB27 des(B28-B30) human insulin and
(Nxcex5B26-tetradecanoyl) LysB26 des(B27-B30) human insulin.
(Nxcex5B32-tetradecanoyl) GluB30, SerB31, LysB32 human insulin.
(Nxcex5B29-acetyl, Nxcex5B32-tetradecanoyl) GluB30, SerB31, LysB32 human insulin.